Microfluidic switching system for analyzing chemotaxis responses of wortmannin-inhibited HL-60 cells

Liu, Yuxin; Sai, Jiqing; Richmond, Ann; Wikswo, John P.

doi:10.1007/s10544-007-9158-z

Cited by 24 publications

(35 citation statements)

References 25 publications

(29 reference statements)

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“…PI3K may not play a critical role in sensing and guiding cells to move in the direction of a chemoattractant gradient. However, when cells experience a change in the direction of the gradient, those cells without PI3K activity lost rapid directional responses to the new gradient (31). It is also interesting to observe that dHL60 cells reversed their polarity, rather than turn in response to the directional change of the CXCL8 gradient.…”

Section: Discussionmentioning

confidence: 97%

Parallel Phosphatidylinositol 3-Kinase (PI3K)-dependent and Src-dependent Pathways Lead to CXCL8-mediated Rac2 Activation and Chemotaxis

Sai

Raman

Liu

et al. 2008

Journal of Biological Chemistry

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The requirement for phosphatidylinositol 3-kinase (PI3K) in the establishment of cell polarity and motility in a number of cell types has recently come into question. In this study, we demonstrate that inhibition of PI3K by wortmannin in neutrophil-like differentiated HL60 cells expressing CXCR2 resulted in reduced cell motility but normal chemotaxis in response to a gradient of CXCL8. However, wortmannin inhibition of PI3K did impair the ability of cells to re-orient their polarity and respond quickly to a change in the direction of the CXCL8 gradient. We hypothesized that Src-regulated ELMO-Dock2-Rac2 activation mediates chemotaxis in the absence of PI3K activity. Inhibition of Src with the small molecule inhibitor, PP2, or inhibition of Dock2 by shRNA knockdown confirmed the functional role of Src and Dock2 in regulating chemotaxis when PI3K was inhibited. Moreover, neutrophils isolated from bone marrow of hck ؊/؊ fgr ؊/؊ lyn ؊/؊ mice exhibited much more severe inhibition of chemotaxis when PI3K was blocked with wortmannin as compared with neutrophils isolated from bone marrow of wild-type mice. Thus, PI3K and Src-ELMO-Dock2 pathways work in parallel to activate Rac2 and modulate chemotaxis in response to a CXCL8 gradient in neutrophils.Chemotaxis is the process by which cells migrate toward a chemical gradient. It has been reported that phosphatidylinositol 3-kinases (PI3K) 2 and their product, phosphatidylinositol 3,4,5-trisphosphate (PIP3), play an important role in sensing and polarizing the cell toward a chemoattractant gradient(1-4). PI3K␥, the dominant class I PI3K in neutrophils, catalyzes the 3Ј-phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate PIP3. In PI3K␥ knockout mice, recruitment of neutrophils to sites of inflammation was significantly reduced, though not completely inhibited. Additionally, in vitro chemotaxis assays showed incomplete inhibition of chemotaxis for neutrophils derived from these mice (5-7). Based on these data, it has been hypothesized that a PI3K-independent pathway exists in neutrophils that allows these cells to move toward a gradient of chemokine. Src family kinases (SFK) are a group of tyrosine kinases that play fundamental roles in mediating cell proliferation, survival, and focal adhesion regulation in response to a variety of environmental stimuli. In certain G protein-coupled receptor-mediated signal transduction pathways, SFK can bind and be activated by G␣ i and G␣ s subunits of the heterotrimeric G-protein (8). This PI3K-independent signal transduction pathway activated directly by heterotrimeric G proteins may lead to a different cascade of signal activation.Members of the Rho family of small GTPases are essential for regulation of cell cytoskeleton reorganization during motility and thus have great impact on cell morphology (polarized versus non-polarized). Activation of Rac has been shown to be required for extension of pseudopodia at the leading edge of moving cells. Rac2, as a hematopoietic-specific member of Rac, plays critical roles i...

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Section: Discussionmentioning

confidence: 97%

Parallel Phosphatidylinositol 3-Kinase (PI3K)-dependent and Src-dependent Pathways Lead to CXCL8-mediated Rac2 Activation and Chemotaxis

Sai

Raman

Liu

et al. 2008

Journal of Biological Chemistry

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“…Therefore, HL-60 cells were stably transfected with a CXCR2 expression vector and the transduced cells exhibited a robust chemotaxis in response to a CXCL8 gradient (Sai, Walker, Wikswo, & Richmond, 2006). The response of these cells to a chemotactic gradient of CXCL8 can be visually observed with a microfluidic device, as described in Liu et al (2008), Sai et al (2006), and Walker et al (2005).…”

Section: Methodsmentioning

confidence: 99%

“…2) (Liu et al, 2008). This device allows one to change the direction of the gradient in less than 1 min and is a very useful tool to study how the cells respond to the directional change of the gradient.…”

Section: Introductionmentioning

confidence: 99%

Study of Chemotaxis and Cell–Cell Interactions in Cancer with Microfluidic Devices

Sai

Rogers

Hockemeyer

et al. 2016

Methods in Enzymology

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Microfluidic devices have very broad applications in biological assays from simple chemotaxis assays to much more complicated 3D bioreactors. In this chapter, we describe the design and methods for performing chemotaxis assays using simple microfluidic chemotaxis chambers. With these devices, using real-time video microscopy we can examine the chemotactic responses of neutrophil-like cells under conditions of varying gradient steepness or flow rate and then utilize software programs to calculate the speed and angles of cell migration as gradient steepness and flow are varied. Considering the shearing force generated on the cells by the constant flow that is required to produce and maintain a stable gradient, the trajectories of the cell migration will reflect the net result of both shear force generated by flow and the chemotactic force resulting from the chemokine gradient. Moreover, the effects of mutations in chemokine receptors or the presence of inhibitors of intracellular signals required for gradient sensing can be evaluated in real time. We also describe a method to monitor intracellular signals required for cells to alter cell polarity in response to an abrupt switch in gradient direction. Lastly, we demonstrate an in vitro method for studying the interactions of human cancer cells with human endothelial cells, fibroblasts, and leukocytes, as well as environmental chemokines and cytokines, using 3D microbioreactors that mimic the in vivo microenvironment.

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“…The ability of flow-based devices to generate concentration gradients of multiple growth factors and the relative stability of the generated gradient are some of the advantages of these devices [93][94][95][96]. Moreover, dynamic control of concentration gradient is also possible within these devices [97][98][99]. However, these devices require fluid convection through the cell culture chamber, which is not appropriate for shearsensitive and non-adherent cells, and can be a confounding factor in many cell culture systems.…”

Section: Developing Novel Microfluidic Devices and Microenvironments mentioning

confidence: 99%

Integrative Utilization of Microenvironments, Biomaterials and Computational Techniques for Advanced Tissue Engineering

Shamloo

Mohammadaliha

Mina

2015

Journal of Biotechnology

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Microfluidic switching system for analyzing chemotaxis responses of wortmannin-inhibited HL-60 cells

Cited by 24 publications

References 25 publications

Parallel Phosphatidylinositol 3-Kinase (PI3K)-dependent and Src-dependent Pathways Lead to CXCL8-mediated Rac2 Activation and Chemotaxis

Parallel Phosphatidylinositol 3-Kinase (PI3K)-dependent and Src-dependent Pathways Lead to CXCL8-mediated Rac2 Activation and Chemotaxis

Study of Chemotaxis and Cell–Cell Interactions in Cancer with Microfluidic Devices

Integrative Utilization of Microenvironments, Biomaterials and Computational Techniques for Advanced Tissue Engineering

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